MYC is the most frequently amplified gene in human cancer and is overexpressed as a consequence of mutations in diverse oncogenic signaling pathways. This proposal will address key questions to resolve the mechanism of MYC transcriptional activation, explore the structure of the unique MYC transactivation domain, and study the impact of mRNA cap methylation which may mediate MYC activities as much or more than transcription. Given the central role of MYC in cancer biology, each of these interrelated goals will offer important new insight into the growth of cancer cells and how this may differ from normal cells. Aim 1: How does MYC activate transcription? The best characterized MYC cofactor is TRRAP, which links MYC to two large acetyltransferase complexes. We will define the genome wide role of this mechanism in MYC-dependent transcriptional activation and identify the substrate(s) of acetylation. Alternatively, we will explore direct MYC-dependent recruitment of the TFIIH and/or PTEFb kinase complexes which regulate RNA polymerase II initiation and elongation. Aim 2: How does MYC enhance mRNA cap methylation? We discovered that MYC can regulate mRNA cap methylation over and above its role in transcriptional activation. This finding has broad ramifications for our understanding of MYC responsive genes since the net effect of increased cap methylation is significantly greater than transcriptional induction for every target gene analyzed. We will use eIF4E-beads to provide a comprehensive assessment of differential cap methylation. We will also define the interactions between MYC, the cap methyltransferase (RNMT) and the RNAPII CTD that are the likely mediators of the activity. Finally, we will explore a broader role for differential cap methylationin other important signaling pathways. Aim 3: What is the structure of the unique MYC transactivation domain? The structure of the DNA binding domain was determined many years ago, but little is known about the structure of the MYC transactivation domain, an unanswered question for most transcription factors. We hypothesize that the transactivation domain may acquire a defined structure in complex with a key transcriptional cofactor, TRRAP. The MYC transactivation domain, especially the essential MBII motif, is highly unique and nearly invariant in evolution and could be a target for specific inhibitors of MYC function, in contrast to the heli-loop-helix/leucine-zipper domains that are shared by over 100 other transcription factors.